生防菌哈茨木霉FJAT-9040的GFP标记及土壤定殖示踪

哈茨木霉Trichoderma harzianum FJAT-9040对茄科尖孢镰刀菌具有较强拮抗作用。为跟踪分析该菌株在土壤中的存活与定殖特性,利用PEG-CaCl2介导的原生质体转化体系,筛选获得1株荧光性状稳定的菌株FJAT-9295,该菌株在生长速率、产孢量、对酸碱度和温度的适应性及对尖孢镰刀菌的抑菌活性等方面与野生型菌株FJAT-9040无显著差异（P〉0.05）。同时,研究了菌株FJAT-9295在4种类型土壤中的定殖能力以及作物生长对该菌株在土壤中定殖的影响。结果表明：菌株在育苗土中定殖最好,其次为沙土及菜园土,黄泥土中定殖最差;种植茄子比未种植作物的土壤更有利于菌株的存活;菌株FJAT-9295在不同类型土壤中的菌落数随时间的延长均略有下降,16天后趋于稳定,维持在105 CFU/g,较初始接菌量下降了约1个数量级。

Transformation of the biocontrol strain FJAT-9040 of Trichoderma harzianum with green fluorescent protein gene and tracing its colonization in soils

A biocontrol fungus FJAT-9040 identified as Trichoderma harzianum, was found to have high inhibition on mycelial growth of Fusarium oxysporum isolated from solanaceous crops. In order to visualize survivability and colonization of this biocontrol fungus in soils, green fluorescence protein gene (gfp) was transformed into the strain FJAT-9040 using protoplast-mediated genetic transformation. A transformant, FJAT-9295, with strong and stable expression of the fluorescent protein was chosen to compare their biological characteristics with the wild type strain FJAT-9040. There were no significant differences between the transformant and the wild type strains in growth rate, spore yield, adaptability to culture pH or temperatures and antifungal activity (P>0.05). The transformant FJAT-9295 was then used to visualize its colonization in four types of soils and the effects of plants on its colonization. Among the four types of soils, the best soil for colonization of the strain FJAT-9295 was nursery soil, the garden soil and sandy soil ranked second, and the last one was loess soil. The population of strain FJAT-9295 in the eggplant-grown soils was higher than that in the non-crop control soils. After mixed with soils, the population of strain FJAT-9295 decreased gradually compared with the initial inoculum in different types of soils; however, they were stabilized with approximately 10⁵ CFU/g after 16 days in the soils.